Microorganisms play an essential role in the recycling of various elements such as the well-established cycles of carbon, nitrogen and sulfur. Moreover they are critical in the biochemical cycles of heavy metals that also occur in the aquatic and terrestrial environment.
Trace metals constitute a significant class of groundwater contaminants, originating from mining effluents, industrial wastewater, landfill leachate, agricultural wastes and fertilizers, and fossil fuels. Based on the chemical properties of dissolved species trace metals can be divided into two distinctive groups: i) reducible metals and metalloids, which are present in natural waters as anions and oxyanions (e.g.; Cr, As, Se, Mo, U), and ii) metal cations, which occur in aqueous environment as divalent cations (e.g.; Cu, Zn, Cd, Pb, Hg, Ni). Depending on their aqueous form, the mobility of trace metals in groundwater is affected by various chemical reactions, including dissolution-precipitation, oxidation-reduction, adsorption-desorption and complexation. Naturally occurring reducing conditions as well as In Situ Chemical Reduction (ISCR) approaches to address the contamination in the subsurface tend to create the phenomenon of biomethylation of the heavy metals. The ISCR remedial technique that was developed over a decade ago uses an array of synergistic interactions between various biotic (e.g., microbial fermentation of organic carbon sources) and abiotic processes (e.g., zero-valent iron [ZVI] chemistry) to encourage dehalogenation of organic compounds as well as reductive precipitation of various heavy metals. Despite the effectiveness of the method, ISCR processes are often confronted by an inability to meet stringent regulatory requirements. One likely explanation for this phenomenon is that, with the possible exception of lead, almost all Group IV, V and VI elements can be biomethylated. The methylmetal(loids) created are usually volatile and more toxic than their inorganic counterparts due to increased water solubility and hydrophobicity.
Microbes have evolved diverse strategies to overcome the toxic effects of metals and metalloids, utilizing accumulation, resistance or, more interestingly, by reducing their bio-availability or toxicity through biomethylation and transformation. The elevated concentrations of waterborne heavy metals have been recognized as an environmental problem in aquatic ecosystems throughout the world. Some of these heavy metals reach the groundwater and others accumulate in seafood or in plants and represent a major toxic source for humans. Some of them cause deformation of birds and sea animals in addition to some diseases in humans.
The synthesis and transfer of methyl groups is an important and widely distributed metabolic process. The following natural products containing one or more methyl groups attached to nitrogen atoms were discovered early in the 19th century: creatine, creatinine, choline, and trimethylamine. Another compound, trimethylglycine ([trimethylammonio]acetate), originally named lycine (from Lycium barbarum) was later renamed betaine (from Beta vulgaris). This discovery contributed the term “betaine” to chemical nomenclature to describe zwitterionic types, for instance: (CH3)nX+—CH2-COO− (if X=N, n=3; if X=S, n=2).
The first observation of a biological methylation came when His, with an interest in the detoxification of aromatic compounds, administered pyridine to a dog. N-Methylpyridine was excreted in the urine. Similar examples of the methylation of aromatic compounds were the conversion, xanthine→methylxanthine in rabbits, and nicotinic acid→trigonelline in dogs.
The role for methionine, a methylated sulfur compound, was also slowly recognized, and S-adenosylmethionine (SAM) was identified as the product of the enzymatic activation of methionine in transmethylation reactions. The role of SAM as the methyl donor in hundreds of methylation reactions is now well established. The mechanism for the de novo biosynthesis of methyl groups has also been determined.
The carbon (C), oxygen (O), nitrogen (N) and sulfur (S) atoms of organic compounds frequently function as methyl group acceptors in primary and secondary metabolic processes. Metalloids specifically tend to be used as methyl group acceptors with a major emphasis on the production of volatile compounds by microorganisms. The term “biomethylation” describes the formation of both volatile and nonvolatile methylated compounds of metals and metalloids.
Biomethylation and hydride generation of group 15 and 16 metals and metalloids (As, Se, Sb, Te, and Bi) by microorganisms are widespread phenomena in anaerobic habitats including landfills, sewage sludge fermentation, alluvial soils, and, as recently shown, the gut of mice and humans.
Microorganisms are primarily responsible for the biosynthesis of organo-metals, and the activity of methanogens is a main source of their production. As Table 1 shows a large number of methanogens have been shown to methylate a variety of metals.
TABLE 1Volatile Methylmetal(loids) produced by Growing Cultures of Methanogens (Archaea)Metal/metalloidAsBiSeTeSbReferenceMethanobacteriumAsH3,CH3AsH2,BiH3, CH3BiH2,(CH3)2Se,(CH3)2TeSbH3CH3SbH2,Michalke etformicicum(CH3)2AsH,(CH3)2BiH,(CH3)2Se2(CH3)3SbH,al., 2000(CH3)3As, X(CH3)3Bi(CH3)3SbMethanobrevibacterCH3AsH2,CH3BiH2,(CH3)2Se,(CH3)2Te(CH3)3SbThis studysmithii(CH3)2AsH,(CH3)2BiH,(CH3)2SeS,(CH3)2As(CH3)3Bi(CH3)2Se, XMethanococcusCH3AsH2,CH3BiH2(CH3)2Se,(CH3)2Te(CH3)3SbThis studyvanielli(CH3)2AsH,(CH3)3Bi(CH3)2SeS(CH3)3As, XMethanolacinian.d.(CH3)2BiH,(CH3)2Se,(CH3)2Te(CH3)3SbThis studypaynteri(CH3)3Bi(CH3)2SeS,(CH3)2Se2, XMethanolobusn.d.(CH3)3Bi(CH3)2Se, X(CH3)2TeCH3SbH2,(CH3)3SbThis studytindariusMethanoplanus(CH3)3As(CH3)3Bi(CH3)2Se,(CH3)2Te, X(CH3)3SbThis studylimicola(CH3)2SeS,(CH3)2Se2, XMethanosarcinaAsH3, X(CH3)3Bi*(CH3)2Se,n.d.(CH3)3SbMichalke etbarkeri(CH3)2Se2al., 2000Methanosarcina(CH3)3As(CH3)3Bi(CH3)2Se,(CH3)2Te(CH3)3SbThis studymazei(CH3)2Se,Methanosphaera(CH3)2AsH,CH3BiH2,(CH3)2Se,(CH3)2Te(CH3)3SbThis studystadtmanae(CH3)3As(CH3)2BiH,(CH3)2SeS,(CH3)3Bi(CH3)2Se2, XMethanothermobacterAsH3n.d.n.d.n.d.(CH3)3SbMichalke etthermautotrophicusal., 2000X, unidentified volatile metal(loids);n.d., not detected;*mediated by addition of octamethylcyclotetrasiloxane and the ionophores lasalocide and monensin.
Methylcobalamin [CH3Cob(III)]-dependent methylation of As, Se, Sb, Te, Hg, and Bi has been reported for numerous anaerobic prokaryotes. In particular, autotrophic sulfate-reducing bacteria as well as methanoarchaea were suggested to be responsible for this process, as CH3Cob(III) and CH3Cob(III)-dependent enzymes are integral parts of physiological pathways such as carbon fixation via the reductive acetyl-coenzyme A (CoA) pathway and methanogenesis. Hence, these organisms contain high concentrations of corrinoids. Nonenzymatic methylation of some metal(loid)s, like As and Hg, by CH3Cob(III) under reductive conditions was assumed by some researchers.
In methanogenesis, the methyl group of CH3Cob(III) is transferred to 2-mercaptoethanesulfonate (CoM) in the penultimate step of methane formation, forming methylated CoM (CH3CoM) and reduced cobalamin [Cob(I)] (18). In the methanol-utilizing methylotrophic pathway, this step is catalyzed by the methyltransferase MtaA (FIG. 1).
As researchers have shown the mechanism for the biomethylation of arsenic has been studied extensively with various fungi, bacteria, archaea, and mammals, including humans. As proposed by Challenger (1945), the biomethylation of selenium and tellurium follows the same mechanism as arsenic. Mercury biomethylation, on the other hand, has been studied in more detail because of poisonings by methylmercury compounds. Additionally the methylated antimony species, trimethylantimony, was detected as a biovolatilization product of antimony in the headspace of soil samples and of pure cultures of Scopulariopsis brevicaulis. The biomethylation of inorganic tin has also been reported, although research on organotin compounds has focused mainly on the fate of organotin species, which are used as biocide additives. These compounds are leached out in aquatic environments and undergo dealkylation and methylation. The methylated bismuth species, trimethylbismuth (TMBi), has been found in gases released from municipal waste deposits and sewage gases, but the origin of this compound is unclear.
Biomethylation of arsenic to trimethylarsine was confirmed in fungi by Frederick Challenger and his co-workers in 1933. FIG. 2 shows the stepwise path involving oxidative addition followed by the reductive elimination that was later proposed for enzymatically catalyzed methylation. Challenger suggested that “active methionine,” later identified as S-adenosylmethionine (SAM), was the methyl group donor. Notably, the Challenger pathway is analogous to the uncatalyzed oxidative addition reaction known as the Meyer reaction that is used to prepare MMA(V) from arsenite and methyl halide. The Challenger pathway can be fully modeled by using the trimethylsulfonium ion as methyl donor and sulfur dioxide as the reducing agent.
Challenger's pathway makes clear predictions about the reaction in which a methyl group is transferred to an arsenic atom, about the charge on the methyl group, and about the oxidation state of the arsenic atom during and after the transfer. The pathway is usually written in terms of oxy-species, but it can be reasonably assured that the As—S bonding plays a major role because of the kinetic stability of the As—S bond to hydrolysis (one of the sources of the well-known affinity of As for S). Electrons for reduction of the methylarsenic(V) species to methylarsenic(III) probably come from oxidation of two thiols to a disulfide as in the real or notional reductive elimination reaction suggested for model systems: R3As(SR′)2→R3As:+R′S—SR′0.16 In enzymatically catalyzed reactions, physiological dithiols such as thioredoxin or glutaredoxin which are reversibly oxidized likely provide these electrons.
FIG. 3 summarizes postulated steps in the methylation of arsenic by the Challenger and alternative pathways. Equation 1 in FIG. 3 shows a variant form of Challenger's pathway in which the As(III) reactant is written as a tris-thiol derivative such as arsenic tris-glutathione. Here, transfer of the electrophile CH3+ from SAM to an As(III) atom yields Sadenosylhomocysteine (SAH), a neutral species, and a methylated arsenical containing an As(V) atom. This is a chemically plausible reaction scheme because there is no possibility of an unfavorable electrostatic interaction between the positive leaving group and the uncharged SAH.
“Reductive methylation”, is the heart of an alternative pathway proposed by other researchers. They suggest that the oxidative methylation and reductive elimination reactions of the Challenger pathway occur simultaneously so that the “real” reaction product is an arsenic(III) species. Therefore, this reaction scheme would significantly reduce the nucleophilicity of the lone electron pair on arsenic and inhibit the reaction.
Other reaction schemes have been suggested based on evidence from studies that used purified arsenic methyltransferases. Two such proteins have been identified and their genes cloned. Arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes arsenic methylation in a wide range of higher organisms, and arsenic methyltransferase (ArsM) catalyzes these reactions in Archaea, some eukaryotes, and many prokaryotes.
Mercury is a naturally occurring element that is found in air, water and soil. It exists in several forms: elemental or metallic mercury, inorganic mercury compounds, and organic mercury compounds. In its elemental form or as inorganic salts, it is relatively less toxic. However, when mercury in these forms is discharged into natural waterways, it is converted into highly toxic and malicious methylmercury ion, CH3Hg+.
The methylation of mercury in water bodies is occurring due to the presence of methanogenic bacteria. Higher levels of methylmercury substances, such as methylmercury chloride are found in deep rather than shallow sediments, indicating that biomethylation largely occurs under anaerobic conditions. The conversion of mercury into its methylated form is fostered by the enrichment of water with organic impurities that permit the growth of methanogenic bacteria. Biomethylation proceeds most effectively in the pH range of 5.5-6.5.
The methylated mercury (MeHg) species monomethyl mercury (MMHg) and dimethylmercury (DMHg) are typically measured at much less than 0.5 pM in aquatic regions, yet MMHg is the form that accumulates in biota. In freshwater and coastal ecosystems the bulk of methylated mercury production is MMHg, thought to be mediated by anaerobic bacteria, particularly sulfatereducing bacteria (SRB), in the redox transition zones of the anoxic layers of the sediment and water column. By analogy, it is thus conceivable that Hg methylation in the open ocean may occur principally in anoxic or sub-oxic zones.
Monomethyl mercury (CH3Hg+, methylmercury, MeHg) is a potent neurotoxic compound. It is biomagnified in the food webs of aquatic systems, reaching high concentrations in carnivorous fish, thus posing a risk to human health. The production of MeHg has been linked to obligate anaerobic bacteria in the δ-Proteobacteria, including iron and sulfate-reducing bacteria (FeRB and SRB) that live in soil and sediments. Although mechanisms of Hg(II) methylation by methylating enzymes have been proposed, the mechanism of Hg(II) uptake by the bacteria has remained obscure. The dominant view is that cellular uptake occurs by passive diffusion of neutral Hg(II) complexes, particularly sulfide complexes, through external membranes, leading to accidental methylation of some of the intracellular Hg(II). However, this view is based on indirect data and modeling, as the precipitation of metal sulfides in the medium and the extensive Hg binding to the surface of the organisms have made it difficult to directly measure Hg(II) uptake in methylating bacteria.
Research on the biological mechanism of mercury methylation has been conducted on one strain of SRB, Desulfovibrio desulfuricans LS. A corrinoid-containing protein was identified as key to mercury methylation capacity in inhibition experiments. From 14C-labeling studies and enzyme activity experiments, researchers concluded that the corrinoid-containing protein responsible for mercury methylation in D. desulfuricans LS is involved in the acetyl-coenzyme A (CoA) pathway.
The acetyl-CoA pathway (FIG. 4) is a carbon metabolism pathway that converts acetate into carbon dioxide (and vice versa), through the breakdown of acetate into carbon monoxide (CO) and a methyl moiety by carbon monoxide dehydrogenase (CODH), and subsequent oxidation of both to CO2. Prior research with Moorella thermoacetica, a non-Hg methylating acetogen, identified a corrinoid-containing protein in the pathway that donated a methyl group to CODH. Presumably, a similar corrinoid protein is involved in the acetyl-CoA pathway in SRB.